The present invention relates to a method for detecting or quantifying an analyte, and a test device used in the method. More particularly, the invention relates to a biosensor test device and method employing marker-loaded liposomes and electrochemical detection for signal amplification and quantitation.
There exist a variety of techniques useful for detecting and/or measuring the concentration of an analyte in a test sample. Such techniques include immunoassays, as described in U.S. Pat. Nos. 5,789,154; 5,756,362; and 5,753,519, each of which is hereby incorporated by reference.
Immunoassays employing electrochemical detection are described in U.S. Pat. No. 4,822,566 to Newman, and Niwa, O.; Xu, Y.; Halsall, H. B.; and Heineman, W. R. Anal Chem. 1993, 65, 1559-1563 (xe2x80x9cNiwaxe2x80x9d). Newman describes a multilayer immunoassay device which relies on the movement of biological species into or out of a biological binding layer in the course of biospecific binding reactions. This movement changes the dielectric constant of the fluid medium containing the analyte, resulting in capacitance changes detected by a sensor. A capacitor comprised of an array of interdigitated copper and gold fingers (2 mil wide, 0.5 mil high, separated by 3 mil spaces) formed by photolithographic etching techniques is disclosed. Niwa describes an electrochemical enzyme immunoassay which employs an interdigitated array microelectrode cell to detect 4-aminophenol (PAP), produced during enzyme immunoassay of mouse IgG. The gold interdigitated array consisted of 50 pair of 3 or 5 xcexcm wide microbands, spaced 2 xcexcm apart. Silver-plated and unplated gold square electrodes were used as reference and auxiliary electrodes, respectively. The assay was conducted in microwells.
The devices and techniques in Newman and Niwa, however, are relatively complex. For example, the enzyme immunoassay described in Niwa is carried out through multiple steps to completion on an immunowell device, and the reaction solution is then transferred to a separate electrochemical detection device.
Nucleic acid detection methods are potentially useful for detecting and measuring the presence of organisms, such as pathogens in food and water supplies. Southern, northern, dot blotting, reverse dot blotting, and electrophoresis are the traditional methods for isolating and visualizing specific sequences of nucleic acids. Each has advantages and disadvantages. For example, gel electrophoresis, often performed using ethidium bromide staining, is a relatively simple method for gaining fragment length information for DNA duplexes. This technique provides no information on nucleotide sequence of the fragments, however, and ethidium bromide is considered very toxic, although safer stains have been developed recently.
If, in addition to length information, there is a desire to determine the presence of specific nucleotide sequences, either Southern blotting, for DNA, or northern blotting, for RNA, may be chosen. These procedures first separate the nucleic acids on a gel and subsequently transfer them to a membrane filter where they are affixed either by baking or UV irradiation. The membrane is typically treated with a pre-hybridization solution, to reduce non-specific binding, before transfer to a solution of reporter probe. Hybridization then takes place between the probe and any sequences to which it is complementary. The initial hybridization is typically carried out under conditions of relatively low stringency, or selectivity, followed by washes of increasing stringency to eliminate non-specifically bound probe and improve the signal-to-noise ratio.
Originally, probes were often labeled with 32P which was detected by exposure of the membrane to photographic film. Today, however, many researchers are making use of non-isotopic reporter probes. These blotting procedures require more time and effort than simple gel electrophoresis, particularly when low levels of nucleic acid are present.
Dot, or slot, blotting are essentially equivalent methods which provide sequence homology information only. No separation of nucleic acid sequences is performed prior to hybridization thus saving considerable time. Typically, the entire DNA, or RNA, composition of the sample being evaluated is attached to a nylon, or nitrocellulose, membrane to form a small xe2x80x9cdotxe2x80x9d of the nucleic acid mixture. The membrane is then probed in a fashion similar to that described for Southern and northern blotting. The technique is simpler than Southern and northern blotting but can give rise to non-specific binding of the probe thus reducing sensitivity. Probes can be labeled with 32P, biotin, various haptens, or enzymes such as horseradish peroxidase and alkaline phosphatase to produce a colored spot on the membrane in the presence of appropriate substrate.
In the reverse dot blot technique, an oligonucleotide capture probe is immobilized on a membrane while the target is kept in solution. In this scheme the target must also bear the reporter entity, usually by indirect registration. An advantage of this strategy is that multiple capture probes can be immobilized on the same membrane so that several target sequences can be determined simultaneously.
There are a wide variety of DNA and RNA detection schemes in the literature, many of which are available as commercial kits. Nucleic acid detection schemes have seen the same trends in assay design as immunoassays, with efforts directed towards simpler, more rapid, and automatable detection schemes.
It is useful to categorize assays based on the fashion in which the signal is produced and detected. Vener et al. (1991) Anal. Biochem. 198, 308-11, classified hybridization probe use into two categories: direct registration and indirect registration. Direct registration, not to be confused with direct hybridization, is defined as the use of a reporter probe which itself is capable of producing a detectable signal. This may be by labeling with a radioisotope, fluorescent tag, enzyme, or sol particle. Most of the initial work done with nucleic acid hybridization made use of direct registration with 32P labelled probes.
An example of this type of assay is that reported by Pollard-Knight et al., (1990) Anal. Biochem. 185, 84-9. These researchers used probes labelled directly with horseradish peroxidase in an enhanced chemiluminescence detection scheme. Single-copy sequences of human genomic DNA were immobilized on nitrocellulose membranes, by Southern blotting, and targeted with enzyme-labelled probes of lengths between 50 and 3571 bases. One enzyme existed for every 50-100 bases of the probe so that better sensitivity was obtained with longer probe lengths. The use of a special blue sensitive film, along with a commercial enzyme substrate, allowed the detection of one amol of several different target sequences.
In indirect registration the probe itself does not bear the signal producing, or reporter entity, rather it bears a ligand such as biotin, fluorescein, digoxigenin, or, in some cases, a non-complimentary nucleotide sequence, which is then specifically recognized by a separate biomolecule or receptor. The latter then either generates or bears the signal producing molecules. This type of assay is very commonly used as a non-isotopic replacement for 32P labeling and is available as commercial kits produced by Amersham International (Arlington Heights, Ill.) and Boehringer Mannheim (Indianapolis, Ind.).
The water-borne pathogen Cryptosporidium parvum illustrates the need for efficient and inexpensive nucleic acid detection methods. Cryptosporidium parvum is found in water supplies and food. Its life cycle includes oocysts that can be difficult to control by drinking water treatment processes such as chemical disinfection and filtration. The ingestion of oocysts can cause serious illness. Table 1 shows a number of documented water-borne outbreaks of Cryptosporidiosis in recent history.
These outbreaks and the difficulty of eliminating Cryptosporidium oocysts from water supplies by conventional water treatment methods demonstrate the need for efficient and inexpensive organism detection methods. Methods for the detection of oocysts have been proposed but they are plagued by poor recovery efficiencies, and seldom provide information regarding the viability of the oocysts that are found in the samples. They also share the deficiencies of existing methods for detecting organisms generally, such as cell culture, agar plate testing, tissue culture, and traditional immunoassay methods, which are laborious, time consuming, and expensive.
In view of the deficiencies and complexities of prior techniques for use as rapid, reliable, and simple assays, the need remains for technology which will accurately detect and determine analytes such as environmental and food contaminants, including pathogenic organisms.
The present invention provides a method and device for detecting or quantifying an analyte in a test sample employing an electrochemical signal production and amplification system. The test device includes a first absorbent material having a contact portion for receipt of the test sample and other assay components. It further includes a capture portion either on said first absorbent material, or on a second absorbent material in fluid flow contact with said first absorbent material. The capture portion has a first binding material bound to the capture portion.
The test device further includes an electrode array having a first conductor and a second conductor. Each conductor comprises a plurality of fingers, and the fingers of the first conductor are interdigitated with the fingers of the second conductor. The electrode array is positioned to induce redox cycling of an electroactive marker released in the capture portion.
The test device is employed in the method of the invention. In the method, the test sample is applied to the contact portion. Either before or after the application of the test sample, it is contacted and with a conjugate of electroactive marker-encapsulating liposomes and a second binding material. The second binding material is selected to bind with a portion of the analyte. The first binding material is selected to bind with a portion of the analyte other than the portion for which the second binding material is selected. The test sample and conjugate are incubated for a time sufficient to permit reaction between any analyte present in the sample and the second binding material.
The test sample is allowed to migrate from the contact portion toward and then into the capture portion. A voltage sufficient to induce redox cycling of the electroactive marker contained in the liposomes is applied across the conductors. After the test sample and the conjugate are incubated, liposomes bound in the capture portion are lysed to release the marker, which undergoes redox cycling as the result of the voltage applied across the conductors, causing current to flow between said first and second conductors. The presence or the amount of the resulting current is detected and correlated with the presence or amount, respectively, of the analyte in the test sample. In this embodiment, the magnitude of the current released from liposomes bound in the capture portion is directly proportional to the amount of analyte in the test sample.
In another embodiment of the invention, the electrode array is positioned to induce redox cycling of electroactive marker released from liposomes which migrate out of the capture portion. Thus, the presence or amount of the current generated by liposomes which are not bound in the capture portion is detected or measured. In this embodiment, the magnitude of the current generated is inversely proportional to the amount of analyte in the sample.
The device and method of the invention can be used directly in the field. The device is used only once, and, therefore, is free from residual environmental contaminants other than what may be present in the sample to be measured. Samples can be assayed within minutes after collection, with the results immediately available on-site. In addition, the device and method of the invention are less complex than many of the prior materials and methods. The ability to deliver quantitative results without additional steps for spectrophotometric or fluorimetric analysis, is an advantage of the present electrochemical device and method over devices and methods that employ dyes and fluorescent materials as markers.
In addition, electroactive marker-loaded liposomes as used in the device and method of the invention provide a highly sensitive, rapid or even instantaneous signal production/amplification system. Furthermore, the amount of marker measured in the electrochemical measurement portion of the absorbent material of the test device is directly proportional to the analyte concentration in the sample. This feature of the invention provides a particular advantage over prior test devices, nucleic acid detection assays, and immunoassays, providing an intuitive correlation between signal strength and analyte concentration. Electrochemical detection offers greater sensitivity than colorimetric determination and is comparable to fluorimetry or chemiluminescence. In addition, the present invention provides quantitative results that can be obtained directly from the electroanalyzer or other detection instrumentation to which the test device is connected, without the need to transfer the device to a separate optical measurement device. Also, electrochemical detection allows for testing in solutions or mixtures that are highly colored or include particulate matter, and which, therefore, would interfere with optical detection.
Interdigitated electrode arrays are particularly suitable for test strip analysis due to their planar configuration and their inherent sensitivity for electrochemical measurements. Microelectrodes fabricated in an interdigitated array have inherent advantages in signal detection over more conventional electrode configurations. These advantages can only be realized with electrodes of very small dimensions due to the theoretical relationships between electrode geometry and ionic diffusion. Scaling down the size of an individual electrode has the advantage of increasing the rate of mass transport, increasing the signal-to-noise (faradaic/charging current) ratio, and decreasing ohmic signal losses, as described in M. Fleischmann, S. Pons, D. R. Rolison, P. P. Schmidt, Eds. Ultramicroelectrodes (Datatech Systems, Inc., Morganton, N.C. 1987), which is hereby incorporated by reference. Advantages of microelectrodes are also described in J. O. Howell, Voltammetric Microelectrodes, Bioanalytical Systems, Inc., West Lafayette Ind. 47906, hereby incorporated by reference.
Advantages of fabricating small electrodes in interdigitated arrays go even further by allowing redox cycling of ions back and forth between anode(s) and cathode(s). See O. Niwa, Y. Xu, B. H. Halsall, W. R. Heineman, Anal. Chem. 65, 1559-1563 (1993) and O. Niwa, H. Tabei, Anal. Chem. 66, 285-289 (1994), each of which is hereby incorporated by reference. This generates much larger currents for detection and allows for the use of extremely small sample volumes. By using a dual potentiostat and a four-electrode system with an interdigitated array, it is possible to almost completely eliminate charging current. This results in a greater signal-to-noise ratio and allows for the use of extremely high scan rates. See O. Niwa, M. Morita, H. Tabei, Anal. Chem. 62, 447-452 (1990) and C. Chidsay, B. J. Feldman, C. Lundgren, R. W. Murray, Anal. Chem. 58, 601-607 (1986), which are hereby incorporated by reference. Furthermore, the sophisticated electronics needed to detect the very small currents associated with individual microelectrode filaments are not necessary due to the summation of current from the large array of microelectrodes.